Some berberine molecule modifications that abrogate its inhibitory effect on cell oxidative phosphorylation.
Authors: Irina V. Mezhevova, Oleg I. Kit, Svetlana Yu Filippova, Anastasia O. Sitkovskaya, Nadezhda V. Gnennaya, Tatiana V. Shamova, Sofia V. Timofeeva, Inna A. Novikova, Yaroslav S. Enin, Oleg N. Burov, Elena Yu Zlatnik, Anna P. Menshenina, Tatiana I. Moiseenko, Ekaterina V. Verenikina, Sergey V. Tumanyan, Nina M. Abdullaeva, Anna A. Cherkasova, Polina A. Kruze, Meri L. Adamyan
Journal: Journal of Clinical Oncology (2022)

e15078 Background: It is known that berberine inhibits oxidative phosphorylation by suppressing respiratory complex I function, but the exact mechanism of this phenomenon has not yet been described. The aim of the study was to evaluate the effect of residues in positions 8 and 13 of berberine molecule on the energy metabolism of HeLa cells. Methods: HeLa cells were seeded in an amount of 2*10 4 cells per well in a Seahorse XFp Analyzer plate (Agilent, USA) in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA). After cell adhesion, the medium was decanted, and the medium with test substances at a concentration of 5 µM was added. Four variants of the experiment, 3 repetitions each, were set up: 1) control; 2) berberine without modification; 3) modification 1 (M1) with the addition of 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl residue at position 13 and malononitrile residue to position 8; 4) modification 2 (M2) with the addition of dicyanovinyl residue at position 13. After 24 hours the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured on Seahorse XFp Analyzer (Agilent, USA) using a Seahorse XFp Cell Energy Phenotype Test (Agilent, USA), with 2 µM FCCP and 1 µM oligomycin. Results: Incubation with the original berberine resulted in a significant decrease in baseline OCR by 39.3±2.8% (M±SD) compared to control (α = 0.05, df = 4), but the maximum OCR did not change significantly. At the same time, a significant increase in the baseline level of glycolysis was observed proportional to the decrease in the baseline OCR, as can be seen from the increase in ECAR by 32.9±5.3% compared with the control (α = 0.05, df = 4). Berberine M1 modification led to a significant increase in baseline OCR by 49.1±2.1% compared with the control (α = 0.05, df = 4), which was also accompanied by a significant increase in maximum OCR by 22.8±3.8% (α = 0.05, df = 4), while the increase in the baseline level of ECAR by 9.7±4.2% was not significant. The addition of the M2 modification did not significantly change the measured parameters compared to the control. Conclusions: The introduction of a dicyanovinyl residue into the berberine molecule at position 13 abrogated its ability to inhibit cellular respiration, possibly due to disturbing its interaction with the proteins of the respiratory complex I. Introduction of the bicyclic residue 7-nitrobenzo[c][1,2,5]oxadiazol-4- yl at position 13 and a malononitrile residue at position 8 caused an increase in baseline and maximum respiration levels, that means thereby berberine activity reversal. This effect can be associated either with the ability to uncouple oxidative phosphorylation (similar to FCCP) or to enhance the expression of electron transport chain proteins.